Università degli Studi di Bologna Alma Mater Studiorum Ph.D. in Plant Pathology - AGR/12 XXIII COURSE Faculty of Agricultural Science Department of Food Improvement and Protection Study of Italian isolates of Alternaria spp.: molecular and morphological characterization and pathogenesis on apple tree CANDIDATE TUTOR: Dott.ssa Francesca Rotondo Prof. Agostino Brunelli COORDINATOR Prof. Paolo Bertolini Keywords: Alternaria spp., phylogenesis, fingerprinting, pathogenesis 2011 1 2 1. Summary In 1999, a symptomatic disease was reported among a number of apple tree orchards (Golden cultivars) in North Italy (Trentino Alto Adige region) (Marshall et al., 2004, and Marshall et al., 2006). By 2002 the disease was found in an area 10 times larger than the initial infection sites and had started attacking the Gala variety of apple, broadening to Veneto, Pidemont (Cuneo and Alessandria) (Ortalda, 2006) and some areas of Emilia Romagna (Ferrara and Ravenna with spots present only on leaf) (Antonacci and Montuschi, 2006). Originally, the disease was attributed to a lack of microelements or a nutritional disequilibrium. In 2003, the Experimental Centre of Laimburg (BZ) demonstrated the pathogenicity of Alternaria isolates on healthy apple foliar tissues and proposed a pathotype of Alternaria alternata as the causal agent of the disease (Gobber et al., 2004 and Marshall et al., 2006). The severe damages inflicted on orchards been the impetus to more deeply investigate the diversity and spatial distribution of Alternaria spp. isolated from these diseased tissues and discover the relationship of the Italian pathogen to the Alternaria alternata mali pathotype, causal agent of the Apple Blotch disease. It was decided to carry out a morphological and molecular characterization of the isolates, evaluating their pathogenicity and, subsequently, combining the collected data to more fully characterize the strains. The strain collection (174 isolates) was constructed by collecting material (received from extension service personnel) between June and August of 2007, 2008, and 2009. All the isolates were sampled from symptomatic tissues (leaves and fruits) from apple orchards in different geographical area in the North of Italy. All the colonies isolated were tested for pathogenicity. Preliminary bioassays were performed on detached plant materials (fruit and leaf wounded and unwounded), belonging to the Golden cultivar, with two different kind of inoculation (conidial suspension and conidial filtrate). Symptoms were monitored daily, and a value of pathogenicity score (P.S.) was assigned on 3 the basis of the diameter of the necrotic area that developed. On the basis of the bioassays, the number of isolates to undergo further molecular analysis was restricted to a representative set of single spore strains (44 strains). Morphological characteristics of the colony and sporulation pattern were determined according to previous systematic work on small-spored Alternaria spp. (Pryor and Michailides, 2002 and Hong et al., 2006). Reference strains (Alternaria alternata, Alternaria tenuissima, Alternaria arborescens and four Japanese strains of Alternaria alternata mali pathotype), used in the study were kindly provided by Prof. Barry Pryor, who allowed open access to his own fungal collection. Molecular characterization was performed combining and comparing different data sets obtained from two distinct molecular approachs: 1) investigation of specific loci and 2) fingerprinting based on diverse randomly selected polymorphic sites of the genome. For the specific locus investigations, it was chosen to sequence the EndoPG partial gene and three anonymous regions (OPA1-3, OPA2-1 and OPa10-2). These markers proved to be a powerful tool in the latter systematic works on small-spored Alternaria spp. It has been reported in the literature that small-spored Alternaria taxonomy is complicated due to the inability to resolve evolutionary relationships among the taxa because of the lack of variability in the markers commonly used in fungal systematics, making this choice of loci appropriate. The three data sets together provided the necessary variation to establish the phylogenetic relationships among the Italian isolates of Alternaria spp. On Italian strains these markers showed a variable number of informative sites (ranging from 7 for EndoPg to 85 for OPA1-3) and the parsimony analysis produced different tree topologies that were all concordant to define A. arborescens as a mophyletic clade. Fingerprinting analysis (nine ISSR primers and eight AFLP primers combination) led to the same result: a monophyleic A. arborescens clade and one clade containing both A. tenuissima and the A. alternata strains. This first attempt to characterize Italian Alternaria 4 species recovered from apple produced concordant results with what was already described in a similar phylogenetic study on pistachio (Pryor and Michailides, 2002), on walnut and hazelnut (Hong et al., 2006), apple (Serdani et al., 2002) and citrus (Peever et al., 2004). Together with these studies, this research demonstrates that the three morphological groups are widely distributed and occupy similar ecological niches. Furthermore, this research suggests that these Alternaria species exhibit a similar infection pattern despite the taxonomic and pathogenic differences. The molecular characterization of the pathogens is a fundamental step to understanding the disease that is spreading in the apple orchards of the north Italy. At the beginning the causal agent was considered as Alteraria alternata (Marshall and Bertagnoll, 2006). Their preliminary studies proposed a pathogenic system related to the synthesis of toxins. Experimental data from our bioassays suggest an analogous hypothesis, considering that symptoms could be induced after inoculating plant material with solely the filtrate from pathogenic strains. Moreover, some positive PCR reactions using AM-toxin gene specific primers, designed for identification of apple infecting Alternaria pathovar, led to a hypothesis that a host specific toxin (toxins) could be involved. It remains an intriguing challenge to discover whether or not if the agent of the “Italian disease” is the same of the one previously typified as Alternaria mali, casual agent of the apple blotch disease. 5 6 2. Introduction 2.1 The genus Alternaria – Historical overview The Alternaria genus is spread worldwide and able to colonize many different habitats, as a parasite on living plant tissue or a saprophite on organic substrata. It is not always easy to distinguish between the two different lifestyles due to the intermediate position of some specimens that are able to shift from the saprophytic to parasitic strategy when faced with a weak host (Rotem, 1994). The genus Alternaria was originally described by Nees in 1817 with A. tenuis as the type and only member of the genus. In 1917 Elliott discussed the generic traits of Alternaria stressing the form of conidia: obclavate, pointed, and often beaked. Then in 1945, Neergaard published his treatise, Alternaria, which was helpful in delineating the main characteristics of the genus but was limited to an accepted 16 species, two varieties, and a few formae speciales known in Denmark. Despite his inclusion of names not always accepted by other taxonomists, the Neergaard treatise was useful in assistance and in identification because it remarked the differences among isolates of the same species. Later, Joly (1964) focused on 28 species, including several Ulocladium taxa within his Alternaria concept. Ellis (1971, 1976) published precise descriptions and drawings of 44 Alternaria taxa focusing, principally, on macro sporulating pathogens. Furthermore, the author performed one of the first descriptions concerning the conidia shape and their morphological characteristics. This was an important milestone even if the key was too simplified, did not give any information regarding media, and did not includes beaks in measuring the conidia dimensions. Recently, Simmons, with his taxonomic essays enclosed in Alternaria Themes and Variations (1981-2003), intended to cover the entire genus with detailed descriptions, drawings, and culture/isolation media and procedures. Exploiting the diversity in spore 7 shape, size, and branching among the Alternaria spp., the author organized the genus into species-groups by referring to a representative species. Most important groups under the pathogenic profile are: the Alternaria alternata group, the Alternaria tenuissima group, the Alternaria infectoria group, the Alternaria arborescens group, the Alternaria brassicicola group, the Alternaria porri group and the Alternaria radicina group (Simmons 1992, Simmons & Roberts 1993, Simmons 1995 and Simmons 1999). Even with the precise descriptions of the differences among various species, these characters remain difficult to exploit by plant pathologists in routine identifications. Because of the absence of an identified sexual stage for the vast majority of Alternaria species, this genus was classified into the division of mitosporic fungi, or the phylum Fungi imperfecti. Latter taxonomic studies have recognized some Alternaria as the anamorphic form of the genuus Lewia, and the current classification reported on Dictionary of Fungi 10th edition (Kirk et al., 2008) is: Phylum: Ascomycota Class: Dothiodeomycetes Subclass:Pleosporomycetes Order: Pleosporales Family: Pleosporaceae Genus: Alternaria (anamorph.) / Lewia (telomorph.) 2.2 The genus Alternaria – Morphological